Morphological, Anatomical Structure, and transcriptomics analysis reveals the regulatory mechanism of cassava plant height development

doi:10.21203/rs.3.rs-4144739/v1

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Morphological, Anatomical Structure, and transcriptomics analysis reveals the regulatory mechanism of cassava plant height development

https://doi.org/10.21203/rs.3.rs-4144739/v1

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published 17 Jul, 2024

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Background: Cassava is one of the three major potato crops in the world, and also the sixth-largest food crop in the world. Improving yield is still one of the main goals of cassava breeding. Plant height has a significant impact on the yield and quality of crops. However, the mechanism of cassava plant height development is still unclear.

Results: In this study, we performed phenotypic, anatomic structural, and transcriptomic analyses to study the mechanisms responsible for Cassava plant-height development. Phenotypic and anatomical observations showed that compared to the high-stem cassava cultivar, the dwarf-stem cassava cultivar displayed a significant decrease in plant height, while the internode tissue xylem area significantly increased. Physiological analysis showed that the lignin content of dwarf cassava was significantly higher than that of high cassava. Transcriptome analysis showed that genes involved in cell wall synthesis and expansion, plant hormone signal transduction, phenylpropanoid biosynthesis, and flavonoid biosynthesis were significantly differentially expressed in the internode tissues of two cassava cultivars.

Conclusion: These results indicate that the internode tissue cell division, secondary wall lignification, and hormones and their related gene expressions play an important role in cassava plant height development. It provides new insights into the mechanism of plant height morphogenesis in cassava. In addition, these candidate genes that regulate plant height development can serve as valuable genetic resources for future crop dwarfing breeding.

Plant height

Cassava

Transcriptome

Physiology

Anatomical Structure

Plant height is one of the important traits that determine crop yield and quality. Crop dwarf breeding is extremely important in production. Plant height dwarfing is conducive to mechanized harvesting, increasing yield and photosynthetic efficiency, fertilizer tolerance, dense planting, lodging resistance, and enhancing the transport of crop nutrients to reproductive organs. In addition, it can also reduce the use of chemical growth regulators, which is conducive to protecting the ecological environment. Therefore, the regulation mechanism and genetic basis of plant height traits have always been a hot topic in genetic breeding research, especially in rice, which has been studied for a long time and systematically. In the 1960s and 1970s, the application of rice and wheat dwarf varieties significantly increased crop yield, which triggered the "Green Revolution"[1].

In addition to the influence of the external environment on plant height, gene regulation plays a leading role in crop plant height development. Han et al. found that the overexpression of BR degradation-related gene AtBAT1 in transgenic creeping bentgrass resulted in plant dwarfing, shorter internodes, smaller leaf angle, and other BR deficiency-related phenotypes[2]. Overexpression of CYP94C2b increased internode length and plant height in rice[3]. The soybean GmLHY gene regulates the plant height development of soybeans by mediating the gibberellin pathway[4]. Overexpression of Rht1 and Rht2 genes in wheat not only greatly reduces the plant height of wheat, but also increases the number of tillers and grains per spike, which greatly improves the yield of wheat[1, 5].

Numerous studies have shown that crop dwarfing is associated with the deletion or mutation of genes involved in gibberellin ( GA ), Brassinosteroid ( BR ), auxin ( IAA ), and strigolactone ( SL ) biosynthesis or signal transduction pathways[6]. Arabidopsis BRI1 (Brassinosteroid-Insenstivie 1), which encodes a receptor-like kinase, whose mutation results in failure to perceive BR signals and leads to an extremely dwarf phenotype[7]. The mutation of GA20ox, a key enzyme in the GA synthesis pathway encoded by the sd1 gene in rice, leads to the decrease of GA content and the shortening of internode length, which makes the plants show a dwarf phenotype[8, 9, 10]. After the deletion of rice auxin co-receptor OsTIR1 and OsAFB2-5 genes, the plant height of rice decreased to varying degrees, accompanied by changes in yield, tillering, root, and other traits[11]. The abnormal expression of the key IAA delivery genes ABCB1/PGP1 in the maize mutant brachytic2 (br2) and sorghum mutant dwarf3 (d3) resulted in dwarfing type[12]. Strigolactone mediates the degradation of D53 by the D14-D3 complex, thereby relieving the repression of downstream target genes and regulating rice plant height[13].

Other factors regulate plant dwarfing traits, such as transcription factors and cell wall-related genes. CesA and CSL genes are responsible for the biosynthesis of the cell wall, and expansin, and XTH genes are involved in the loosening of a cell wall, greatly affecting cell growth during stem elongation[14]. CesA or CSL mutations cause plants to exhibit dwarf phenotypes[15, 16, 17, 18, 19]. In inhibition of expansin gene expression, plants showed reduced plant height, early flowering, and leaf curl[20, 21, 22, 23]. Transcription factors can respond to external environmental factors and endogenous hormone signals, and promote plant growth by regulating downstream target genes[24, 25, 26]. For example, overexpression of NAC transcription factors reduced the content of abscisic acid and BR, resulting in shorter internodes in apples [27]. Although significant progress has been made in the genetic regulation mechanism of crop height, and some genes related to plant height have been identified, plant height is a complex quantitative trait controlled by multiple genes, and the functions and regulatory mechanisms of many genes are still unclear. Therefore, more research is needed to explore plant height genes to fully understand their role in plant height regulation.

Cassava (Manihot esculenta Crantz ) is a plant of the genus Manihot in the family Euphorbiaceae and is listed as the world's three major tuber crops along with potato and sweet potato. With an annual output of more than 200 million tons, it is the staple food for the survival of more than 700 million people around the world. It is listed as the sixth-largest food crop in the world. It is also an important industrial raw material, feed, biomass energy, and famine relief crop. It plays a very important role in world food security, biomass energy, and food processing[28, 29]. It has a reputation as the ' king of starch ' and ' energy crop '. Cassava is resistant to barren, dry, and acidic soil and can be planted on slope land. It is mainly asexual reproduction, with high photosynthetic efficiency, extensive management, low cost, high yield potential, and many other advantages, and has great industrial development potential. However, the research on cassava plant height development has not been reported. Therefore, in this study, two cassava varieties with significant differences in plant height were used as materials, we analyzed and discussed the formation mechanisms of cassava plant height development based on morphological structure, anatomical structure, lignin content, cellulose content, and gene expression. The results of this study revealed the molecular mechanism of cassava plant height development and provided an effective theoretical basis for cassava dwarf breeding.

Comparison of the phenotypic characteristics, cytology, and physiology of the XX048 and NZ199 plants

The plant height of the two varieties of XX048 and NZ199 was 420.27 ± 5.2cm and 219 ± 4.57cm, respectively, and the difference was extremely significant (Fig. 1). August to September is the period of rapid growth in cassava plant height. During this period, the height of XX048 plants increased by nearly 150cm, while NZ199 plants increased by about 55cm, XX048 plants grew faster than NZ199 plants, and the difference in height between the two was the largest during this period.

To observe the anatomical differences between XX048 and NZ199, we performed paraffin section staining in the stem of the top internode tissue. The woody area of XX048 is 414.87 ± 14.28mm², while that of NZ199 is 911.10 ± 82.62mm², with significant differences between the two. The average length and width of XX048 xylem cells were 27.04 ± 4.37mm and 22.11 ± 4.36mm, respectively, while the average length and width of NZ199 xylem cells were 35.25 ± 3.81mm and 29.12 ± 5.15mm(Fig. 2), respectively. There was no significant difference in length and width between XX048 and NZ199. These results indicate that the xylem of NZ199 is more developed and has a higher degree of lignification.

The content of lignin and cellulose in the stem of the top internode tissue of XX048 and NZ199 is shown in Fig. 3. The lignin content of NZ199 and XX048 is 162.37 ± 1.81mg/g and 132.01 ± 3.83mg/g, respectively. The cellulose content of NZ199 and XX048 is 73.47 ± 2.69mg/g and 70.91 ± 1.45mg/g, respectively. The difference in lignin content between the two is significant, while the difference in cellulose content between the two is not significant. The results showed that the cell wall lignification development of the two was different, and the main stem of NZ199 cassava entered the secondary wall lignification development state earlier. This result is consistent with the cytological results.

Transcriptomic analysis

To explore the key genes related to plant height development of cassava, six cDNA libraries were constructed. Based on Illumina sequencing, after removing splices and low-quality reads, 121791451 clean readings were obtained(Supplementary Table 1), and the percentages of Q20, and Q30 base rates were more than 97.82% and 93.65%, respectively. The clean reads of each sample were compared with the cassava reference genome, and the comparison efficiency ranged from 94.56–94.88%. The sample correlation heat map showed that the R² value among three biological repetitive samples was greater than 0.89, and that of most of the samples was greater than 0.9, indicating that this experiment was highly repeatable and the data were reliable (Fig. 4).

To study the expression pattern of genes related to plant height development of cassava, the transcriptome map of cassava with tall and short stems was compared, and the differentially expressed genes of cassava with tall and short stems were identified by log2 | Fold Change | ≥ 2 and p - value < 0.01. A total of 2368 DEGs were differentially expressed between XX048 and NZ199, of which 1112 genes were up-regulated and 1256 genes were down-regulated (Fig. 5).

To infer the biological processes of all DEGs, We have classified DEGs into GO functions, the identified DEGs were assigned into three main GO functional categories(Fig. 6), including biological process(BP), cellular component(CC), and molecular function(MF). The three most abundant sub-categories in which the BP category was divided were “metabolic process”(753 DEGs), “cellular process”(590 DEGs), and “single-organism process” (458 DEGs). For the CC category, the three most abundant sub-categories were “membrane”(521 DEGs), “membrane part” (480 DEGs), “cell part” (377 DEGs), and “cell” (377 DEGs). The majority of DEGs of the MF category were assigned to “binding” (829 DEGs), “catalytic activity”(795 DEGs), and “transporter activity”(105 DEGs).

The KEGG enrichment analysis found that the five most abundant pathways as "Plant-pathogen interaction" (117 DEGs), “Plant hormone signal transduction" (47 DEGs), "Starch and sucrose metabolism" (46 DEGs), "phenylpropanoid biosynthesis" (43 DEGs), and "Carbon metabolism (41)". In addition, the “Flavonoid biosynthesis”, “Brassinosteroid biosynthesis”, and “Zeatin biosynthesis”, pathways were also enriched, and the “Flavonoid biosynthesis” pathway was the most significantly enriched(Fig. 7).

DEGs involved in plant hormone biosynthesis and signal transduction pathways

Plant hormone biosynthesis and signal transduction pathways include auxin, gibberellin, cytokinin, BL, abscisic acid, ethylene, jasmonic acid, and salicylic acid synthesis and transduction. Many important genes involved in auxin, cytokinin, gibberellin, and BL biosynthesis and signal transduction pathways were significantly down-regulated in dwarf cassava (Fig. 8, Supplementary Table 2). In the IAA biosynthesis and transduction pathway, auxin response factor ( ARF ), auxin-responsive GH3 gene family ( GH3 ), and small auxin-up RNA(SAUR) were generally down-regulated. In the cytokinin pathway, two-component response regulators ARR-A and ARR-B families were generally down-regulated. In the GA pathway, one DELLA protein was up-regulated, one was down-regulated, phytochrome-interacting factor 3 ( TF ) was down-regulated, and gibberellin receptor GID1 ( GID1 ) was up-regulated. Several BL biosynthesis and signal transduction genes were also down-regulated, including brassinosteroid insensitive 1-associated receptor kinase 1 ( BAK1 ), protein brassinosteroid insensitive 1 ( BRI1 ), and cyclin D3 ( CYCD3 ). Many important genes involved in ETH, SA, JA, and ABA biosynthesis and signal transduction pathways were significantly up-regulated in dwarf cassava. In the ABA pathway, serine / threonine-protein kinase SRK2 ( SnRK2 ) and ABA-responsive element binding factor ( ABF ) were generally up-regulated. In the JA pathway, transcription factor MYC2 (MYC2) was generally up-regulated. In the ETH and SA pathway, serine / threonine-protein kinase CTR1 ( CTR1 ), mitogen-activated protein kinase kinase 4 / 5 ( MKK4 _ 5 ), regulatory protein NPR1 ( NPR1), pathogenesis-related protein 1 ( PR1 ) were up-regulated.

DEGs involved in flavonoid biosynthesis and phenylpropanoid biosynthesis

The flavonoid biosynthesis pathway was the most significantly enriched in the KEGG enrichment analysis. In the flavonoid biosynthesis pathway, most of the genes were significantly up-regulated in the XX048 Vs.NZ199 group, including 4 chalcone synthase genes EC: 2.3.1.74 ( CHS ), 5 shikimate O-hydroxycinnamoyltransferase EC: 2.3.1.133 ( HCT ). 1 phlorizin synthase genes EC: 2.4.1.357 ( PGT1 ), 2 chalcone isomerase EC: 5.5.1.6, 1 naringenin 3-dioxygenase EC: 1.14.11.9 ( F3H ), 1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase EC:1.1.1.219 1.1.1.234 (DFR), 1 flavonoid 3',5'-hydroxylase EC:1.14.14.81 (CYP75A), 1 flavonol synthase EC:1.14.20.6(FLS), 1 anthocyanidin synthase EC:1.14.20.4 (ANS),1 anthocyanidin reductase EC:1.3.1.77(ANR), 1 leucoanthocyanidin reductase EC:1.17.1.3(LAR). Only 1 chalcone synthase gene EC: 2.3.1.74 ( CHS ), 1 caffeoyl-CoA O-methyltransferase gene EC: 2.1.1.104, and 5 phlorizin synthase genes EC: 2.4.1.357 ( PGT1 ) were significantly down-regulated in the XX048 Vs.NZ199 group(Fig. 9, Supplementary Table 3).

The phenylpropanoid biosynthesis pathway is also one of the KEGG enrichment pathways. The products of the phenylpropanoid biosynthesis pathway include lignin monomers such as p-coumarol, coniferyl alcohol, and sinapyl alcohol. It showed 37 significantly differentially expressed genes involved in lignin monomer synthesis (Fig. 10, Supplementary Table 4), including 4 phenylalanine ammonia-lyase ( PAL ), 6 4-coumarate-CoA ligase ( 4CL ), 5 cinnamoyl-CoA reductase ( CCR ), 4 cinnamyl-alcohol dehydrogenase ( CAD ), 2 peroxidases ( POD ), 5 shikimate O-hydroxycinnamoyltransferase ( HCT ), 5 caffeic acid 3-O-methyltransferase / acetylserotonin O-methyltransferase ( COMT ), 1 caffeoyl-CoA O-methyltransferase ( CCoAOMT ), 1 ferulate-5-hydroxylase ( F5H ) and 4 laccase genes. Twenty-eight genes were significantly up-regulated in the XX048 Vs.NZ199 group and only 9 genes were significantly down-regulated.

DEGs involved in cell wall biosynthesis and expansion

To provide mechanical strength to newly elongated cell walls and internodes, the synthesis of new cell wall components is essential. We analyzed the differential expression of genes involved in cell wall synthesis or modification, such as cellulose synthase ( CES ), expansin ( EXP ), xylosidase ( XYL ), pectin lyase ( PL ), pectin esterase ( PM), pectin acetyl esterase ( PA ) and polygalacturonase ( PG ), which may be involved in the elongation and expansion of cassava stems.

The relaxation of the cell wall is a physiological process that must occur during cell expansion and elongation during plant growth and development. Expansins (EXPs) are nonhydrolytic cell wall-loosening proteins that enable cells to expand while allowing tissues to differentiate and grow. Two members of the expansin gene family were differentially expressed in

tall and dwarf cassava and were significantly up-regulated in dwarf cassava(Fig. 11, Supplementary Table 5 ). Xyloglucan endoglycosyltransferase / hydrolase ( XTH ) has also been shown to participate in cell expansion by relaxing and rearranging cell wall fibers in growing tissues. Four XTHs were differentially expressed between tall and dwarf cassava, of which 3 XTHs were significantly down-regulated and 1 XTH was up-regulated in the tall vs. dwarf group(Fig. 11, Supplementary Table 5 ). Pectin is a complex polymer, and its degradation is the initiation of secondary wall lignification and cell wall disintegration. A total of 11 related genes were significantly differentially expressed in tall and dwarf cassava, including 5 pectin esterase genes ( PM ), 3 polygalacturonase genes ( PG ), 2 pectin lyase genes ( PL ), 1 pectin acetyl esterase gene ( PA ). In addition, 1 cellulose synthase gene and 2 XYL xylosidase genes were also differentially expressed in the tall vs. dwarf group(Fig. 11, Supplementary Table 5 ).

Transcription factor analysis

Transcription factors play an important role in gene expression. In this study, we identified 92 transcription factors differentially expressed in XX048 Vs.NZ199, representing 34 transcription factor families, of which 56 were down-regulated and 36 were up-regulated(Supplementary Table 6). These transcription factors mainly included bHLH ( 9 ), MYB ( 9 ), C2H2 ( 7 ), NAC ( 5 ), MYB-related ( 5 ), bZIP ( 5 ), AP2 / ERF-ERF ( 5 ) and WRKY ( 4 ) transcription factor families.

qRT-PCR validation

To verify the accuracy of RNA-Seq results, 11 differentially expressed genes were randomly selected for qRT-PCR verification. The results showed that the expression patterns of these 11 differentially expressed genes were similar to RNA-Seq, with R² of 0.9804(Fig. 12), which shows that RNA-Seq is reliable.

Plant height is an important agronomic trait affecting crop yield and quality. Understanding the molecular mechanism of controlling crop plant height is of great significance for improving crop yield and quality. However, as one of the world's three major potatoes and six major food crops, the genetic regulation mechanism of cassava plant height is not yet clear, which is not conducive to the regulation and genetic improvement of cassava plant height traits. Therefore, this study aims to identify candidate genes and gene expression networks that regulate cassava plant height development.

The biosynthesis and expansion of cell walls play a major role in plant height

Plant cell wall not only provides mechanical support for cell components, and protects plants from various external stimuli, but also is essential for cell growth. Lignin and cellulose are two important components of cell walls, and their content is the main factor affecting plant height. Studies have shown that the higher the plant height, the lower the lignin content[30]. The change in lignin content is related to the expression of genes related to the lignin biosynthesis pathway. Among them, PAL, HCT, 4CL, CCR, and CAD genes play an extremely important regulatory role in the synthesis of lignin, and the amount of lignin synthesis is positively correlated with the expression of these genes[31, 32]. In this study, a large number of PAL, HCT, 4CL, CCR, and CAD genes were significantly up-regulated expression in dwarf cassava(Fig. 10), which was consistent with the physiological results. The lignin content in dwarf cassava was significantly higher than that in high-stem cassava(Fig. 3). Similar results were also observed in rice, dwarf rice mutants showed higher lignin content and higher lignin-related gene expression levels in stems. In addition, peroxidase and laccase are also involved in the biosynthesis of lignin, and they can catalyze the cross-linking between phenolic groups in cell wall structural proteins, pectin, and other wall polymers, thereby causing cell wall hardening, inhibiting cell elongation, and achieving the goal of cell wall lignification. In this study, the peroxidase gene and laccase gene were significantly up-regulated in dwarf cassava(Fig. 10). The above results indicate that the dwarf cassava has a stronger ability to synthesize lignin macromolecules, indicating that the internode tissue of the dwarf cassava stem has stopped growing and began to develop into a state of lignification. These results indicate that the development of cell wall lignification has a significant impact on plant height during the internode development of cassava.

Expansion is a kind of non-hydrolytic cell wall relaxation protein in plant cell walls. It is considered to play an important role in regulating cell wall ductility and is a key factor in determining cell expansion[22]. Overexpression of OsEXP4 in transgenic rice significantly increased the plant height[23]. Overexpression of ClEXPA2 in tobacco also significantly increased the internode length[33]. Although expansins have long been thought to play an active role in plant height and tissue expansion, this is not the case. Rochange et al.showed that the growth of transgenic plants overexpressing expansin was hindered, manifested as smaller leaves and shorter internodes[34]. Further analysis showed that the appearance of this phenotype was due to the reduced sensitivity of the cell wall to expansin. Interestingly, a similar phenomenon was also observed in this study, two expansin genes were significantly up-regulated in dwarf cassava, indicating that the expansins themselves can be transcribed normally(Fig. 11). Therefore, it is speculated that the decrease of cell wall sensitivity to expansin is one of the main factors affecting the plant height dwarfing of cassava.

XTH encodes a cell wall modifying enzyme found in higher plant cells, which changes the structure of the cell wall by catalyzing the hydrolysis or transfer of xyloglucan molecules. Studies have shown that XTH is closely related to plant height development. In rice, the expression of OsXTH8 was positively correlated with plant height[35]. Mutations in AtXTH9 result in shorter internodes length in Arabidopsis[36]. In this study, the expression level of the XTH genes in high-stem cassava was significantly higher than that in dwarf-stem cassava(Fig. 11), indicating that these genes may be involved in the development of cassava plant height.

The importance of plant hormones in determining plant height

As a physiological signal regulating plant growth and development, plant hormones play an important role in plant morphogenesis, growth, and metabolism. Studies have shown that plant dwarfing mutations are closely related to plant hormones.

Auxin is an important growth regulator and plays an important role in all stages of plant growth and development. Abnormal expression of auxin transport key genes can lead to plant dwarfing. In this study, it was observed that the auxin response gene CH3 ( Manes.12G087200.v7.0 ) was significantly down-regulated in dwarf cassava, indicating that the auxin response was reduced. ARF transcription factors can mediate auxin responses and modulate stalk development by regulating cell division[37]. In dwarf cassava, the expression of ARF was significantly down-regulated(Fig. 8), indicating that the cell division of dwarf cassava stalk was inhibited. SAUR gene is the largest family of auxin response genes, the gain-of-function and loss-of-function SAUR19-24 seedlings exhibit increased and reduced hypocotyl length and leaf size[38]. In this study, a large number of SAUR genes were significantly down-regulated in dwarf cassava(Fig. 8), and similar situations were observed in Sophora davidii and Peanut[39, 40]. The above results indicate that these genes affect plant height during cassava growth and development.

GAs have an important effect on the elongation of plant stalks. The plant hormone GA, the receptor GID1, and the inhibitor DELLA together constitute the GA signal transduction pathway After being activated by GA, the gibberellin receptor GID1 binds to DELLA through the E3 ubiquitination pathway, leading to the degradation of DELLA protein, thereby alleviating DELLA-mediated growth inhibition[41]. The loss-of-function mutation of the GID1 gene resulted in a dwarf phenotype in the plant[42]. Interestingly, in this study, GID1 was significantly up-regulated in dwarf cassava(Fig. 8). This is contrary to the fact that loss-of-function mutations in GID1 cause plants to exhibit dwarf phenotypes and GA insensitivity[42]. However, our finding is in agreement with the GA-triggered negative feedback loop that represses the transcription of GID1 genes in Lepidium sativum[43]. There have been similar situations in previous studies on bamboo. PeGID1 showed higher expression levels in the internodes of dwarf Moso bamboo than that of wild-type[44]. DELLA protein is a transcription regulatory factor responsive to GA and an inhibitory factor for GA-induced growth. The DELLA protein gene PhSLR1 of moso bamboo was transferred into Arabidopsis thaliana, and it was found that the transgenic plants were shorter than the wild type[45]. In this study, we obtained a conclusion similar to previous studies that one DELLA protein gene was up-regulated in dwarf cassava(Fig. 8). Cassava plant height dwarfing may be because the DELLA protein inhibits gibberellin signal transduction, resulting in plant dwarfing.

BRs are important positive regulators of stem elongation. The deletion or mutation of genes, transcription factors, and enzymes related to BR synthesis and signal transduction pathways affect the elongation of plant cells and make plants show a dwarf phenotype. BRI1 is the main component of BR signal perception. Mutants bri1-4 and Osbri1 are dwarfs in Arabidopsis and rice[46, 47, 48]. In addition, BAK1 also plays an important role in BR signal transduction. bak1 loss-of-function mutants express a semi-dwarf phenotype with rounded leaves, shortened stems, and decreased sensitivity to BR that resembles a pheno-typically weak bri1 mutant[49]. CYCD3 is a D-type plant cyclin gene that can promote plant cell division. When CYCD3 was inhibited, Arabidopsis showed significant dwarfing[50]. In this study, 6 BRI1s, 3 BAK1s, and 1 CYCD3 genes were down-regulated in dwarf cassava(Fig. 8), indicating that the sensitivity of dwarf cassava to BR is reduced, and the inhibition of cell elongation may be one of the important factors of cassava dwarfing.

The importance of flavonoids in determining plant height

In recent years, many studies have shown that plant height development is related to flavonoids. Transcriptome and metabolome analysis showed that a series of genes involved in the flavonoid biosynthesis pathway were differentially expressed in dwarf and tall mutants of Sophora davidii, a total of 8 differential metabolites were identified in dwarf and tall mutants, of which 6 were flavonoids, indicating that flavonoid metabolites play an important role in plant height morphogenesis of Sophora davidii[39]. The dwarfing mechanism of Polish dwarf wheat was analyzed by transcriptome and proteome, the results showed that the genes involved in flavonoid synthesis were up-regulated in dwarf wheat, which increased the content of flavonoids in dwarf wheat, thus limiting the transport of auxin, reducing the ductility of cells, and ultimately limiting the expansion of cells, resulting in dwarfing of wheat[51]. It can be seen that flavonoids can regulate plant height development by controlling the polar transport of auxin. Similar to previous studies in the present study, DEGs were analyzed using the KEGG database, the results showed that the flavonoid biosynthesis pathway was the most significantly enriched, and a series of genes involved in the flavonoid biosynthesis pathway ( F3H, CHS, FLS, HCT, and F3′5′H ) were significantly up-regulated in dwarf cassava(Fig. 9). The up-regulated expression of these genes increased the flavonoid content of dwarf cassava, thereby inhibiting the polar transport of auxin, which is essential for the regulation of growth processes such as cell division and cell expansion[52, 53]. Polar transport of auxin is inhibited and cellular extensibility is reduced, limiting cell expansion ultimately leading to dwarfing of the cassava plant height, however, the exact mechanisms involved need to be further investigated.

The important role of transcription factors in plant height development

The transcription factor family plays an important role in regulating the elongation and expansion of plant cells. Some transcription factors have been shown to play critical roles in plant height development, including GRF, PRE, bZIP, TCP, WRKY, etc. In this study, we identified a total of 92 transcription factors differentially expressed in XX048 Vs.NZ199, of which bHLHs and MYBs were the major represented TF families. Many studies have found that bZIP-like transcription factors play an important role in plant growth and development, including seed maturation, germination flower development, etc. In addition, bZIP proteins are also involved in the growth and development of plant height. In Arabidopsis, the bZIP transcription factor affects plant height growth and development by controlling gibberellin activity[54, 55]. In this study, most of the bZIP transcription factors were significantly down-regulated in dwarf cassava, and only one bZIP transcription factor was up-regulated. It was speculated that these down-regulated bZIP transcription factors inhibited the activity of gibberellin, resulting in cassava dwarfing. MYB transcription factors are a large group of transcription factors in plants that participate in various physiological and biochemical processes, including root development, cell development, secondary cell wall synthesis, and seed development. In rice, overexpression of OsMPH1 increased plant height by increasing the length of internode cells[56]. In addition, MYB transcription factors are also involved in plant hormone signal transduction. MYB33 mediates GA signal transduction during plant growth and is a positive regulator of GA signal transduction[57]. In this study, four MYB transcription factors were significantly down-regulated in dwarf cassava, which may inhibit the elongation of internode cells, resulting in cassava dwarfing. bHLH is involved in a series of processes of plant growth and development, including seed germination, floral organ development, and stem and lateral root growth. In Arabidopsis, the mutation of bHLH transcription factor SPATULA limits cell proliferation and expansion, resulting in shortened internode length[58, 59]. The maize double mutants(blh12/14) have early internode differentiation and abnormal branching leads to dwarfism and reduced stem veins[60]. In this study, most of the bHLH transcription factors were significantly down-regulated in dwarf cassava, which may inhibit cell proliferation and expansion, resulting in plant height dwarfing.

Regulatory networks associated with height development in cassava

The plant height development of cassava is a complex regulatory process, which is affected by many factors. Based on the results of this study and previous studies, we proposed a regulatory network model related to cassava plant height development ( Fig. 13 ): As mentioned above, cell wall lignification, decreased expression levels of plant hormone signal transduction-related genes, and excessive accumulation of flavonoid compounds are important reasons for cassava plant height dwarfing. In addition, the down-regulated expression of important genes involved in cell wall synthesis and expansion and some related transcriptional regulatory factors may also affect the height development of cassava. In detail, the expression of genes related to the hormone signaling pathway and cell wall synthesis was down-regulated, which inhibited the biosynthesis and expansion of the cell wall. In addition, some transcription factors are involved in cell development by participating in plant hormone signal transduction. The up-regulated expression of some key enzymes in the lignin synthesis pathway promotes cell wall lignification, prevents cell elongation and growth, and causes dwarfing. The up-regulated expression of flavonoid biosynthesis-related genes promotes the accumulation of flavonoid compounds, thereby inhibiting the transport of auxin, affecting cell division and expansion, and leading to plant height dwarfing. In summary, these differentially expressed genes play an important role in the regulatory network of cassava plant height development, and further functional identification studies are needed to determine the function of these candidate genes.

This study analyzed transcriptome, physiology, and anatomy data to identify candidate genes related to cassava plant height development, and proposed a genetic regulatory network model for cassava plant height development. The molecular mechanisms underlying the influence of plant hormone signaling, transcription factors, cell wall synthesis and enlargement, flavonoid biosynthesis, and cell wall lignification on the development of cassava plant height have been revealed. It is expected to provide a theoretical basis for elucidating the molecular regulatory mechanism of cassava plant height development and improving cassava plant type.

Plant materials, growth conditions, and measurements of agronomic traits

In this study, industrial cassava varieties xinxuan048 (XX048: high-stem) and Nanzhi199 (NZ199:dwarf-stem) were used as experimental materials. All materials were planted and grown in the test base of Guangxi South Asia Tropical Agricultural Science Research Institute in Longzhou County, Chongzuo City, China, under similar conditions. This test base altitude is 138m, which belongs to the southern subtropical monsoon climate. The average annual temperature is ≥ 23.1°C, the annual rainfall is ≥ 1266 mm, the annual sunshine hours are about 1521h, the average annual relative humidity is about 88%, and the annual frost-free period is 361 days. The test base is loam soil with medium fertility. Planted in April 2022. A completely randomized block design was adopted. Each variety was treated as one treatment and was repeated three times, with a distance of 1m between rows and 0.8m between individuals. Field management followed standard agricultural practice.

Two cassava lines XX048 and NZ199 with uniform growth were selected as the research objects to measure the plant height in the stem growth period. The stem of the topmost internode tissue was collected for cytological observation When the plant height difference between XX048 and NZ199 was the largest ( September 2022: tuberous root expansion period ), comparing the microstructure of cassava from XX048 and NZ199, measuring the content of cellulose and lignin, the main components of the cell wall, and conducting transcriptome sequencing comparison.

Cytological observation

In September 2022 (The plant height difference between XX048 and NZ199 was the largest during this period), Randomly selected 5 plants with consistent growth in each planting plot, and stems of the topmost internodes tissues of XX048 and NZ199 were collected respectively. These samples were named XX048-1, XX048-2, XX048-3, NZ199-1, NZ199-2 and NZ199-3. These samples were immediately fixed in formalin-aceto-alcohol (FAA) for more than 24 hours. Before dehydration, plant lignocellulose was softened with an acidic softening solution for several days to a week, and dehydrated with gradient ethanol ( 75%, 85%, 90%, 95%, 100%, 100% ) in turn ( 4h, 2h, 2h, 2h, 2h, 2h, 2h), followed by infiltration with alcohol benzene, the transparent agent I and transparent agent II for 30 min, respectively, and paraffin embedding. 8µm serial sections were cut with 340E (ThermoFisher Scientific, China) and fixed on slides. The sections were stained with toluidine blue, dehydrated and transparent, sealed with neutral gum, and observed with an Olympus BX53F2 photomicroscope (Olympus, Tokyo, Japan).

Determination of lignin and cellulose content

In September 2022 (The plant height difference between XX048 and NZ199 was the largest during this period), Randomly select 5 plants with consistent growth in each planting plot, and stems of the topmost internodes tissues of XX048 and NZ199 were collected respectively. The collected samples were dried to constant weight at 80 ℃, crushed, sieved through 40 mesh, and set aside. Lignin and cellulose content was determined by kit ( Suzhou Keming Biotechnology Co. Ltd., China). The experimental steps were carried out according to the instructions.

Sample collection, RNA extraction, RNA sequencing, and data analysis

The plant's total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the instructions provided by the manufacturer. RNA concentration and purity were measured using NanoDrop 2000(Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).

A total amount of 1µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using Hieff NGS Ultima Dual-mode mRNA Library Prep Kit for Illumina (Yeasen Biotechnology (Shanghai) Co., Ltd.) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First-strand cDNA was synthesized and second-strand cDNA synthesis was subsequently performed. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ends of DNA fragments, the NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Then 3µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37℃ for 15 min followed by 5 min at 95℃ before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Clean reads were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. At the same time, Q20, Q30, GC-content, and sequence duplication levels of the clean data were calculated. All the downstream analyses were based on clean data with high quality. These clean reads were then mapped to the cassava genome sequence. Quantification of gene expression levels was estimated by fragments per kilobase of transcript per million fragments mapped. Genes with an adjusted P-value < 0.01 and | log2 (FoldChange) | > 1 obtained by DESeq2 were considered DEGs. Gene function was annotated based on the following databases: NR (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), KO (KEGG Ortholog database), GO (Gene Ontology).

Fluorescence Quantitative Verification

Real-time quantitative polymerase chain reaction ( qPCR ) was performed as described by Cai et al.[61].The primer sequence is shown in supplementary table S7. The relative expression level was calculated by the 2 ^{− ΔΔ}Ct method with actin as an endogenous reference gene.

BR: Brassinosteroid ; GA: Gibberellin; IAA: Auxin; SL: strigolactone; SA: Salicylicacid; JA: jasmonic acid; ABA: abscisic acid; XTH: xyloglucan endotransglucosylase/hydrolases ;CesA: cellulose synthase; CSL: Cellulose synthase-like; CYCD3: cyclin D3; SnRK2:serine / threonine-protein kinase SRK2; ETH: ethylene; CHS: chalcone synthase; HCT: shikimate O-hydroxycinnamoyltransferase; PGT1 : phlorizin synthase; F3H: naringenin 3-dioxygenase;DFR: bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase; CYP75A: flavonoid 3',5'-hydroxylase; ANS: anthocyanidin synthase; ANR: anthocyanidin reductase; LAR: leucoanthocyanidin reductase; PAL: phenylalanine ammonia-lyase; 4CL:4-coumarate--CoA ligase; CCR: cinnamoyl-CoA reductase; CAD: cinnamyl-alcohol dehydrogenase; POD: peroxidase; HCT: shikimate O-hydroxycinnamoyltransferase; COMT: caffeic acid 3-O-methyltransferase / acetylserotonin O-methyltransferase; CCoAOMT: caffeoyl-CoA O-methyltransferase; CES: cellulose synthase; EXP: expansin; XYL: xylosidase; PL: pectin lyase; PM:pectin esterase; PA: pectin acetyl esterase; PG: polygalacturonase; XTH: Xyloglucan endoglycosyltransferase / hydrolase.

Acknowledgements

Not applicable.

Authors' contribution

HRL and LFH have designed experiments and revised the manuscript. ZQC, LXR, WLW, WH, HYY, and HXC have analyzed the sequencing data for transcriptome assembly. ZHL, ZLH, XL, XFZ, and RLH supported the materials. CHZ and TYL have developed qPCR experiments. ZQC has written the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China(2023YFD1600601), the Guangxi Natural Science Foundation Program of China (2023GXNSFBA026150), the Basic Research Business Special Project of Guangxi Academy of Agricultural Sciences of China(Guinongke 2021YT157)

Availability of data and materials

The materials of this study were provided by the Guangxi South Subtropical Agricultural Science Research Institute. Correspondence and requests for materials should be addressed to Hengrui Li([email protected]). The raw sequencing data have been submitted to the NCBI SRA database (PRJNA976406).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interest.

Method declaration

All methods are implemented in accordance with relevant guidelines and regulations in this manuscript.

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Morphological, Anatomical Structure, and transcriptomics analysis reveals the regulatory mechanism of cassava plant height development

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Journal Publication

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Abstract

Figures

Introduction

Result

Comparison of the phenotypic characteristics, cytology, and physiology of the XX048 and NZ199 plants

Transcriptomic analysis

DEGs involved in plant hormone biosynthesis and signal transduction pathways

DEGs involved in flavonoid biosynthesis and phenylpropanoid biosynthesis

DEGs involved in cell wall biosynthesis and expansion

Transcription factor analysis

qRT-PCR validation

Discussion

The biosynthesis and expansion of cell walls play a major role in plant height

The importance of plant hormones in determining plant height

The importance of flavonoids in determining plant height

The important role of transcription factors in plant height development

Regulatory networks associated with height development in cassava

Conclusion

Materials and Methods

Plant materials, growth conditions, and measurements of agronomic traits

Cytological observation

Determination of lignin and cellulose content

Sample collection, RNA extraction, RNA sequencing, and data analysis

Fluorescence Quantitative Verification

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1